The transcriptional machinery of HIV-1 remains a therapeutic target that has not been exploited in current therapeutic regimens. HIV-1 transcription is uniquely dependent on the action of the viral transactivator Tat. Tat acts as an adaptor protein and recruits important cellular cofactors to the HIV-1 promoter located in the 5-prime LTR. We have recently identified the class III deacetylase SIRT1 as a novel Tat-interacting protein. SIRT1 is a close homologue of the Sir2 protein, a central regulator of longevity and aging in yeast. The goal of this proposal is to study the role of SIRT1 in HIV-1 infection. We have shown that Tat transactivation of the HIV-1 promoter requires the SIRT1 deacetylase activity. Tat and SIRT1 coimmunoprecipitate and synergistically activate the HIV-1 promoter in transformed cell lines. We will now study the role of SIRT1 in HIV-1 infection in primary CD4+ T cells and macrophages, the natural targets of HIV-1. We will introduce specific siRNAs directed against SIRT1 into these cells by nucleofection and study their effect on HIV-1 replication. We will also examine the expression and activity of SIRT1 in primary HIV-1 target cells and perform a comprehensive mutagenesis analysis of SIRT1 to delineate the mechanisms of SIRT1 activity in HIV-1 infection. We will further study the molecular mechanism of SIRT1 action during HIV-1 infection. SIRT1 predominantly targets nonhistone proteins for deacetylation, including the p65 subunit of NF-KB and the Tat protein itself. Using ChIP assays and infectious HIV-1 mutated in the SIRT1 deacetylation site in Tat or mutated in the NF-KB-binding sites in the viral LTR, we will study the relative contribution of Tat and p65 to the regulatory role of SIRT1 during HIV-1 infection. We will also characterize the new interaction of SIRT1 with the class I histone deacetylase HDAC3 and identify transcriptional targets of the SIRT1 deacetylase activity in HIV-1 target cells. We have shown that a structural derivative of splitomicin, a known inhibitor of the yeast Sir2 protein, suppresses Tat transactivation in micromolar concentrations. In collaboration with Elixir Pharmaceuticals Inc. we have now obtained novel specific inhibitors of human SIRT1 that are active in nanomolar concentrations. We will study the effect of these inhibitors in replication studies in primary HIV-1 target cells and cell culture models of HIV-1 latency. These studies should conclusively demonstrate whether SIRT1 is an important cellular cofactor for HIV-1 infection and a potential drug target in infected individuals.